The cloning and expression of varius heme proteins in bacteria has previously been described. Thus, S. A. Ortlepp et al., J. Biotechn. 11, 1989, pp. 353-364, describe the expression and characterisation of horseradish peroxidase C in E. coli. The enzyme is expressed intracellularly as an insoluble aggregate so that it has to be purified from lysed cells. Furthermore, the enzyme is not expressed in active form and must be folded separately in the presence of heme and Ca.sup.2+ to become functional. Similarly, A. T. Smith et al., J. Biol. Chem. 265(22), 1990, pp. 13335-13343, describe the expression of horseradish peroxidase C in E. coli. The recombinant enzyme has less activity than native horseradish peroxidase C and is produced in a yield of 2-3% (of the purified, active enzyme). S. Loprasert et al., J. Bact. 171(9), 1989, pp. 4871-4875, report the cloning and expression in E. coli of peroxidase A from Bacillus stearothermophilus. S. J. Hoffman et al., Proc. Natl. Acad. Sci. USA 87, pp. 8521-8525, describe the expression of functional human hemoglobin in E. coli. Z. Wang et al., J. Biotechn. 13, 1990, pp. 131-144, describe the cloning and expression of lignin peroxidase from Streptomyces viridosporus in Streptomyces lividans.
Expression of human hemoglobins in yeast (Saccharomvces cerevisiae) has been described by M. Wagenbach et al, Bio/Technology 9, 1991, pp. 57-61. In yeast, hemoglobin is expressed as a fully assembled, heme-containing tetramer. However, the protein is not secreted from the yeast cells, but remains in the cytoplasmic space and must be purified therefrom.
It would therefore be advantageous to select a host organism, such as a filamentous fungus, which is capable not only of producing heme proteins but also of exporting them through the cell membrane in active form, thereby simplifying purification procedures.
In recent years, procedures have been developed for the transformation of filamentous fungi, including Aspergillus niger and Aspergillus nidulans. U.S. Pat. No. 4,885,249 (Allelix) describes a general process for the transformation of A. niger, exemplified by the introduction of plasmids carrying genes encoding selectable markers. EP 215 594 (Genencor) describes the expression and secretion of various proteins in A. nidulans, using the signal sequences of different Aspergillus proteins to provide secretion.
Neither of these references indicates the possibility of producing heme proteins in filamentous fungi. On the contrary, M. Saloheimo et al., Gene 85, 1989, pp. 343-351, describe the cloning and expression of a lignin peroxidase from Phlebia radiata in Trichoderma reesei. The authors report that although lignin peroxidase mRNA is expressed in T. reesei, no protein product could be detected. They speculate that this might be ascribable to intracellular degradation by proteases due to incorrect folding of the protein in the absence of heme or to a different structure of the RNA interfering with its translation.